CTHRC1+ fibroblasts are stimulated by macrophage‐secreted SPP1 to induce excessive collagen deposition in keloids

Dear Editor, Fibrosis is characterized by fibroblast dysfunction and excessive deposition of cell-matrix that affect the normal functioning of the original tissue or organ.1 The pathogenesis of keloids is complex and remains elusive. Multiple studies have suggested that skin color and tension,2 immunity,3 hormones, inflammatory stimulation,4 genes5 and other factors play critical roles in the onset and development of keloids. In addition to fibroblasts, immune cells that include macrophages,6 mast cells7 and lymphocytes8 are also important to keloid pathogenesis via cytokine secretion and the triggering of abnormal signaling pathways.9,10 Therefore, performing single-cell RNA-sEquation (scRNA-seq) that can identify heterogenous cell populations and cellular developmental pathways is thus valuable, as it will provide insight into the key pathogenic cell-types and the molecular patterns. In this study, we performed scRNA-seq on six pairs of keloid samples to elucidate the pathogenesis of skin fibrosis. Detailed methods and single-cell sequencing analysis are described in the Supplemental materials. We obtained lesion and non-lesion skin biopsies from six keloid patients (Figure 1A, Figure S1A,B, Table S1) and performed hematoxylin and eosin (H&E) and Masson staining (Figure 1B). Then we applied single-cell sequencing (scRNA-seq) to 12 skin samples that included both lesion and non-lesion areas (Figure 1C). After stringent quality-control procedures, 60,732 high-quality cells were obtained for further analysis. We first visualized all subgroups by uniform manifold approximation and projection (UMAP) and then determined the different cell types that included fibroblasts, keratinocytes, endothelial cells, T cells, pericytes, myeloid cells, mast cells, B cells, adipocytes, melanocytes and salivary gland cells (Figure 1C,D). All cell types were identified in each sample, and we observed minimal batch effects in our study (Figure S1C-–F). Top-marked signatures for each cell type

Dear Editor, Fibrosis is characterized by fibroblast dysfunction and excessive deposition of cell-matrix that affect the normal functioning of the original tissue or organ. 1 The pathogenesis of keloids is complex and remains elusive. Multiple studies have suggested that skin color and tension, 2 immunity, 3 hormones, inflammatory stimulation, 4 genes 5 and other factors play critical roles in the onset and development of keloids. In addition to fibroblasts, immune cells that include macrophages, 6 mast cells 7 and lymphocytes 8 are also important to keloid pathogenesis via cytokine secretion and the triggering of abnormal signaling pathways. 9,10 Therefore, performing single-cell RNA-sEquation (scRNA-seq) that can identify heterogenous cell populations and cellular developmental pathways is thus valuable, as it will provide insight into the key pathogenic cell-types and the molecular patterns.
In this study, we performed scRNA-seq on six pairs of keloid samples to elucidate the pathogenesis of skin fibrosis. Detailed methods and single-cell sequencing analysis are described in the Supplemental materials.
We obtained lesion and non-lesion skin biopsies from six keloid patients ( Figure 1A, Figure S1A,B, Table S1) and performed hematoxylin and eosin (H&E) and Masson staining ( Figure 1B). Then we applied single-cell sequencing (scRNA-seq) to 12 skin samples that included both lesion and non-lesion areas ( Figure 1C). After stringent quality-control procedures, 60,732 high-quality cells were obtained for further analysis. We first visualized all subgroups by uniform manifold approximation and projection (UMAP) and then determined the different cell types that included fibroblasts, keratinocytes, endothelial cells, T cells, pericytes, myeloid cells, mast cells, B cells, adipocytes, melanocytes and salivary gland cells ( Figure 1C,D). All cell types were identified in each sample, and we observed minimal batch effects in our study ( Figure S1C showed unique transcriptomic patterns in accordance with their physical functions ( Figure 1E,F). We noted that extracellular matrix protein (ECM) genes were significantly enriched in fibroblasts, endothelial cells and pericytesindicating an important role for these cell types in keloids ( Figure S1G).
Fibroblasts are considered the primary source of ECM in keloids. We applied UMAP analysis to all fibroblasts ( Figure 2A). Based on their expression profiles, we classified fibroblasts into five subgroups: F01-CCL19, F02-CTHRC1, F03-APCDDA, F04-FGFBP2 and F05-TNN ( Figure 2B, Figure S2A). Intriguingly, ECM gene modules were significantly activated in F02 relative to other genes, as exemplified by CTHRC1 ( Figure 2C); and dualfluorescence staining also confirmed that CTHRC1 was coexpressed with COL1A1/α-SMA ( Figure 2D, Figure S2B). To further substantiate that CTHRC1+ fibroblasts comprised the main source of collagen production, we separated CTHRC1+ cells from the fibroblasts (CD90+) of another three skin samples and found that they exhibited higher expression of COL1A1 and COL1A2 compared to the other genes ( Figure 2E, Figure S2C). Furthermore, when we performed trajectory analysis to elucidate the dynamic relationships among fibroblast subgroups, our data suggested that F01 differentiated into F02 and F03 ( Figure 2F), while F04 and F05 extended minimally into the trajectory ( Figure S2D). The expression of ECM-related genes was gradually augmented in the transition from F01 to F02 fibroblasts, further confirming the role of F02 fibroblasts in collagen production and keloid pathogenesis ( Figure S2E).
In addition, we demonstrated that F02-CTHRC1 fibroblasts were highly enriched in the lesion samples based on the ratio of observed-to-expected cell numbers (Ro/e) ( Figure S3A), which was further validated by IHC staining ( Figure S3B). Thereafter, we performed differential gene expression (DGE) analysis and uncovered several ECMrelated genes as significantly over-expressed in lesion     Table S2). Gene set variation analysis (GSVA) enrichment analysis showed that hedgehog signaling, epithelial mesenchymal transition (EMT) signaling and transforming growth factor (TGF)-β signaling were activated in F02 fibroblasts, but not others, from lesion samples ( Figure S3E); evaluation of gene-module expression revealed that the EMT and the TGF-β-signaling pathways were significantly elevated in the lesion regions ( Figure S3F). Trajectory analysis showed that two key genes, THY1 (CD90) and CTHRC1, were gradually activated in pseudotime from non-lesion to lesion regions ( Figure S3G,H).
To clarify the factors regulating CTHRC1+ fibroblasts, we implemented SCENIC analysis and found that multiple transcription factors (TFs) were activated in CTHRC1+ fibroblasts, including GATA2, SOX7, JDP2, IRF4 and CREB3L1 (Figures S4A and S5A). Through regulon specificity score (RSS) score and module analysis, we determined the M9 module that is comprised of CREB3L1, NR1D1, JDP2, MYLK and CREB5 might be key to the alterations observed in CTHRC1+ fibroblasts (Figures S4B--G and S5A--D). To further illustrate the regulation of TFs, we performed both siCREB3L1 and inhibitor (SGC-CBP300 targets CREBBP/EP300) experiments in primary fibroblasts separated from the lesion regions. We found that the down-regulation of CTHRC1 protein levels in the siCREB3L1 group was coordinated by CREB3L1, which can significantly attenuate fibrosis ( Figure 2G).
Immune cells infiltrate into lesion regions to participate in skin wound healing ( Figure 3A,B, Figure S6A,B). To examine the heterogeneity of macrophages in keloids, we re-clustered macrophages and identified three cell types: Mon-S100A8, Mac-C1QA and Mac-APOC1 ( Figure 3C,D, Figure S6C,D). We found that most Mac-APOC1 (SPP1 high) cells came from lesion regions and that the Mac-C1QA cells came from lesion regions expressing COL1A1 ( Figure 3E,F), where SPP1 and APOE are secreted to cooperate with fibroblasts ( Figure 3G, Figure S6E). We further stimulated fibroblasts with SPP1 in vitro and showed that it enhanced collagen-related protein expression of fibroblasts (α-SMA, COL1A1) in a dose-dependent manner and that it increased the proportions of CTHRC1+ fibroblasts (i.e., CTHRC1 and CREB3L1) ( Figure 3H, Figure S6F).
When we investigated the interactions among all the cell types, we ascertained that monocyte/macrophage cells, endothelial cells, pericytes and fibroblast cells interacted more robustly than the other cell types ( Figure 4A--C). Fibroblasts interacted with endothelial cells via ACKR1, while multiple chemokines (CXCL2, CCL3L3) secreted by myeloid cells interacted with DPP4 in fibroblasts ( Figure 4D).
As described above, we demonstrated a major contribution by CTHRC1+ fibroblasts in collagen deposition and that macrophages infiltrate into the lesion regions; we thus inferred that macrophages communicated with CTHRC1+ fibroblasts to further enhance collagen deposition. We then focused on the ECM pathway in CTHRC1+ fibroblasts and found that the SPP1 that was principally produced by Mac-APOC1 increased ECM expression from CTHRC1+ fibroblasts. Collectively, our results reveal that CTHRC1+ fibroblasts occupy a vital role in excessive collagen deposition forming a positive loop with myeloid cells, endothelial cells and pericytes in this pathologic phenomenon, which carries the potential to become a targeted cell-type in treatment strategies ( Figure 4E).

C O N F L I C T O F I N T E R E S T
All authors declared no conflict of interest.